Throughout this application, various publications are referenced. The disclosures of these publications in their entireties, are hereby incorporated by reference into this application, in order to more fully describe the state of the art, as known to those skilled therein, as of the date of invention, described and claimed herein.
This invention relates to modified recombinant adenovirus (Ad) vectors which lack a gene or genes for replication of the adenovirus in a non-actively cycling cell but remain capable of specifically replicating in an actively cycling cells. These modified recombinant vectors are useful for regulating transgene expression in cells such as tumor cells and therefore, for treatment of a variety of cancers.
Adenovirus Replication
Numerous studies in cell-free systems and in infected cells have established that Ad DNA replication takes place in two steps.
In the first step, DNA synthesis is initiated by the precursor to the terminal protein (pTP). pTP binds as a heterodimer with the Ad polymerase (Pol) to specific sites within the inverted terminal repeat (ITR) sequences. Ad DNA replication begins at both ends of the linear genome resulting in a daughter strand that is synthesized in the 5xe2x80x2 to 3xe2x80x2 direction displacing the parental strand with the same polarity.
Three non-exclusive mechanisms are proposed for the second stage, the replication of the displaced parental strand: i) displaced single strands can form partial duplexes by base pairing of the ITRs on which a second round of DNA synthesis may be initiated, ii) when two oppositely moving displacement forks meet, the two parental strands will no longer be held together and will separate, resulting in partially duplex and partially single-stranded (ss) molecules; synthesis is then completed on the displaced parental strands, and iii) displaced strands with opposite polarity resulting from initiation at two different molecular ends, can renature to form a double-stranded daughter molecule. Replication of DNA requires two components: DBP and polymerase. DBP is thought to stabilize the formation of the panhandle structure and the interstrand renaturation process. Polymerase synthesizes Ad DNA at a rate of 20-30 base pairs (bp) per second.
Ad DNA replication is a crucial prerequisite in Ad recombination. Suppressing Ad DNA replication diminishes Ad recombination.
Adenovirus Recombination
Ad DNA replication generates large amounts of stabilized ssDNA that has been shown to efficiently induce homologous recombination in bacteria. Electron microscopy (EM) studies demonstrated that the displaced parental single strand can be transferred to another duplex genome forming Holliday structures very similar to the structure described for homologous recombination in bacteria.
Early studies focused on Ad recombination revealed that Ad genomes can recombine until late in the infection cycle and each viral genome can undergo multiple recombination events. Population genetics of Ad genomes suggested a model similar to that proposed for T-even phages. More sophisticated studies analyzing cross-exchange of specific restriction sites demonstrated that recombination between Ad genomes can be detected only after the onset of viral DNA replication and that the degree of replication is proportional to the recombination frequency. Suppressing viral DNA replication unambiguously diminishes recombination.
Recombinant Ad vectors are widely used for gene transfer in vitro and in vivo (Hitt, M. M. et al., 1997. Advances in Pharmacology. 40:137-205). First-generation AdE1xe2x88x92 vectors used for gene transfer deleted the E1A/E1B genes and are, therefore, considered replication deficient in normal cells (Jones, N., and T. Shenk. 1979. Proc Natl Acad Sci U S A. 76:3665-9).
The proteins encoded within the early region 1A (E1A-12S and E1A-13S) and 1B (E1B-55k and -19k) mediate viral replication by transactivating viral gene expression and deregulating the cell cycle [for review: (Shenk, T. 1996. Adenoviridea, p. 2111-2148. In B. N. Fields, Knipe, D. M., Howley, P. M. (ed.), Fields Virology, vol. 2. Lippincott-Raven Publisher, Philadelphia)]. E1A-12S and 13S are the first proteins expressed from the incoming viral genome and function as the main transactivators for other Ad transcription units, particularly the E2 and E4 regions that encode proteins for Ad DNA replication. E1A deregulates the cell cycle and induces cellular DNA synthesis by directly transactivating cellular genes [(e.g. c-myc (Hiebert, S. W. et al., 1989. Proc Natl Acad Sci U S A. 86:3594-8), cdc2 (Kao, C. Y. et al., 1999. J Biol Chem. 274:23043-51), hsp70 (Lum, L. S. et al., 1992. Mol Cell Biol. 12:2599-605)], as well as by interacting with cell cycle regulatory proteins like pRb, pRb-related proteins (p107/p130), or p300 (Tiainen, M. et al., 1996. Cell Growth Differ. 7:1039-50). Binding of E1A to pRb-family members releases transcription factors of the E2F family resulting in transcriptional upregulation of host genes; these genes are regulators or effectors of DNA synthesis [e.g. c-myc, Elf-1, myoD, DNA polymerase a, cyclins A and E, PCNA (Dyson, N., 1998. Genes Dev. 12:2245-62)] which eventually drive quiescent cells into S-phase.
These and other cellular factors, whose activities peak during S-phase [e.g. the synthesis of deoxyribonucleotides (Bjorklund, S. et al., 1990. Biochemistry. 29:5452-8; Engstrom, Y. et al., 1985. J Biol Chem. 260:9114-6)], create an environment appropriate for viral DNA synthesis. However, in normal cells, E1A induced cell cycle deregulation results in the accumulation of p53, which stimulates p53 mediated G1 arrest (el-Deiry, W. S. et al., 993. Cell. 75:817-25; Xiong, Y. et al., 1993. Nature. 366:701-4) or apoptotic pathways (Miyashita, T., and J. C. Reed. 1995. Cell. 80:293-9). Notably, E1A induced apoptosis can also occur through p53 independent pathways (Teodoro, J. G. et al., 1995. Oncogene. 11:467-74).
Among the E1B proteins, the 19 kDa and 55 kDa proteins have been shown to cooperate with the E1A-13S product to initiate viral replication by down regulating p53 driven expression of cyclin D1; this is required for cells to progress through the G1 phase of the cell cycle (Spitkovsky, D. et al., 1995. Oncogene. 10:2421-5). Another important function of the E1B proteins is to oppose p53-mediated apoptosis induced by E1A-12S. The E1B 55k protein in concert with E4-orf6 and E4-orf3 directly blocks p53, whereas E1B-19k appears to inhibit a p53-dependent, apoptotic downstream event similar to bcl-2 (Debbas, M. and E. White. 1993. Genes Dev. 7:546-54; Rao, L. et al., 1992. Proc Natl Acad Sci U S A. 89:7742-6). At late time points during lytic infection, E1B complexes to the E4orf6 protein, thereby enhancing the export of viral mRNA from the nucleus while, at the same time, inhibiting the transport of cellular mRNA (Babiss, L. E. and H. S. Ginsberg. 1984. J Virol. 50:202-12; Ornelles, D. A., and T. Shenk. 1991. J Virol. 65:424-9). E1B-55k inactivation of p53 has been hypothesized to be required for productive (wild type) adenovirus replication resulting in lytic infection (Bischoff, J. R. et al., 1996. Science. 274:373-6). Consequently, it has been suggested that an E1B-55k mutant virus should replicate preferentially in p53 deficient tumor cells (Heise, C. et al., 1997. Nat Med. 3:639-45; Heise, C. C. et al., 1999. Cancer Res. 59:2623-8; Kirn, D. H., and F. McCormick. 1996. Mol Med Today. 2:519-27; Wildner, O. et al., 1999. Cancer Res. 59:410-3). However, a number of reports have discredited this idea by demonstrating that the ability of the E1B-55k mutant to productively replicate does not correlate with cellular p53 status (Goodrum, F. D., and D. A. Ornelles. 1998. J Virol. 72:9479-90; Hall, A. R. et al., 1998. Nat Med. 4:1068-72; Harada, J. N., and A. J. Berk. 1999. J Virol. 73:5333-44; Hay, J. G. et al., 1999. Hum Gene Ther. 10:579-90; Rothmann, T. et al., 1998. J Virol. 72:9470-8; Turnell, A. S. et al., 1999. J Virol. 73:2074-83; Vollmer, C. M. et al., 1999. Cancer Res. 59:4369-74).
Loss of p53 function is coincident with the development of a variety of tumors mostly due to accumulation of mutations and the inability to carry out p53 dependent apoptosis (Lowe, S. W. et al., 1994. Science. 266:807-10). Another common defect observed in many different tumors is the loss of control of the G1 to S transition in the cell cycle due to mutation or inactivation of pRb or p16, a kinase inhibitor which regulates the phosphorylation and activity of pRb (Kamb, A. et al., 1994. Science. 264:436-40; Okamoto, A. et al., 1994. Proc Natl Acad Sci U S A. 91:11045-9; Vousden, K. H. 1995. Semin Cancer Biol. 6:109-16). Since AdE1A/E1B inactivate p53 and pRb functions, tumor cell lines with deregulated cell cycles and deregulated p53 and/or pRb pathways may support viral replication in the absence of E1A and E1B.
First generation, E1A/E1B-deleted adenoviruses are used for gene therapy based on the premise that deleting of E1A and E1B sequences should be sufficient to eliminate viral gene expression and replication (Berk, A. J. et al., 1979. Cell. 17:935-44; Hitt, M. M. et al., 1997. Advances in Pharmacology. 40:137-205; Jones, N., and T. Shenk. 1979. Proc Natl Acad Sci U S A. 76:3665-9). However, a large number of studies have demonstrated the expression of viral early and late proteins in cells transduced with E1 deleted AdE1 vectors in vitro and in vivo (Engelhardt, J. F. et al., 1994. Hum Gene Ther. 5:1217-29; Engelhardt, J. F. et al., 1993. Hum Gene Ther. 4:759-69; Jane, S. M. et al., 1998. Ann Med. 30:413-5; Lieber, A. et al., 1996. J Virol 70:8944-60; Yang, Y. et al., 1994. Proc Natl Acad Sci U S A. 91:4407-11; Yang, Y. et al., 1996. J Virol. 70:7209-12). These findings are in agreement with observations made two decades ago that transcription of early Ad genes occurs slowly in the absence of E1A in a dose dependent manner (Gaynor, R. B., and A. J. Berk. 1983. Cell. 33:683-93; Nevins, J. R. 1981. Cell. 26:213-20). It was hypothesized that cellular proteins can substitute functionally for E1A in the transactivation of viral promoters (Gaynor, R. B., and A. J. Berk. 1983. Cell. 33:683-93; Lieber, A. et al., 1996. J Virol. 70:8944-60). Therefore, the first generation Ad E1A/E1Bxe2x88x92 adenoviruses are inadequate gene therapy vehicles.
The present invention is an improvement upon the first generation vectors in specifically targeting cells, such as tumor cells, where Ad DNA replication can occur. Ad DNA replication is only initiated upon the production of a critical threshold of early viral proteins which, in turn, is directly dependent on the number of viral genomes present in nuclei of infected cells [(Shenk, T. 1996. Adenoviridea, p. 2111-2148. In B. N. Fields, Knipe, D. M., Howley, P. M. (ed.), Fields Virology, vol. 2. Lippincott-Raven Publisher, Philadelphia; van der Vliet. 1995. Adenovirus DNA replication., p. 1-31. In a. P. B. W. Doerfler (ed.), The molecular repertoire of adenoviruses, vol. 2. Springer Verlag, Berlin)]. In previous studies, it was demonstrated that de novo DNA synthesis occurs in a number of cell lines and primary cells (including murine hepatocytes) after infection with E1-deleted vectors (Lieber, A. et al., 1996. J Virol. 70:8944-60; Nelson, J. E., and M. A. Kay. 1997. J Virol 71:8902-7). However, AdE1xe2x88x92 viral DNA replication was not detectable in vivo in mouse livers (Nelson, J. E., and M. A. Kay. 1997. J Virol. 71:8902-7). This discrepancy, between primary hepatocytes in vitro and in vivo, prompted us to perform detailed replication studies with E1-deleted vectors in a variety of tumor cell lines. Specifically, we examined the roles of infectivity, tumor suppressor gene status, cell cycling, and presence of serum on vector DNA replication.
Based on the discussion above, a remaining problem in tumor gene therapy involves the inefficient administration of recombinant adenoviral vectors. Efficient adenoviral gene therapy for cancer depends not only on primary gene transfer to a portion of tumor cells, but also on efficient replication and dissemination of the vectors to the surrounding tumor tissue, in order to achieve a complete killing. The invention also addresses this issue.
This invention provides modified recombinant Ad vectors (e.g., first generation-vectors) undergoing defined homologous recombination in order to create predictably rearranged genomic derivatives in a host cell. Genomic rearrangements can be achieved, for example, by incorporating two inverted repeat (IR) sequences within one vector genome and enabling genomic rearrangement by coinfection with two parental vectors of one type (also referred to herein as a one vector system) (see e.g., Example 1) or by homologous recombination of overlapping regions in two distinct types of parental vectors (with or without IR sequences) and enabling genomic rearrangement only upon coinfection of the host cell with the two distinct parental vectors (also referred to herein as a two vector system) (see e.g., Example 2).
The Ad vectors of the invention allow for tumor-specific gene expression through a recombination mechanism, that is based on the selective replication of the vectors of the invention in tumor cells, as disclosed herein.
The invention also provides methods for viral spread. In the tumor-specific vectors of the invention, viral spread may be facilitated by induction of apoptosis, thereby enhancing virus release from infected cells, infection of surrounding tumor cells and increased tumor cell lysis.